Highly potent antibodies binding to death receptor 4

ABSTRACT

The present invention is directed toward monoclonal antibodies that bind to death receptor 4 and/or death receptor 5, a pharmaceutical composition comprising same, and methods of treatment comprising administering such a pharmaceutical composition to a patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a US national stage of PCT/US16/59517 filedOct. 28, 2016, which claims the benefit of provisional application No.62/248,782 filed Oct. 30, 2015, incorporated by reference in itsentirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named512440_SEQLST.txt, created Sep. 9, 2020, and containing 42,000 bytes,which is hereby incorporated by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates generally to the combination of monoclonalantibody (mAb) and recombinant DNA technologies for developing novelbiologics, and more particularly, for example, to the production ofmonoclonal antibodies that bind and activate death receptors 4 and 5.

BACKGROUND OF THE INVENTION

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL, alsoknown as Apo2 ligand, and also designated as Apo2L or Apo2L/TRAIL) is amember of the TNF ligand superfamily (reviewed in IAM van Roosmalen etal., Biochem Pharmacol 91:447-456, 2014). TRAIL is expressed on manycells of the immune system in a stimulus dependent manner and modulatesimmune responses, for example as a key effector molecule in NK cellmediated cytotoxicity (C Falschlehner et al., Immunol 127:145-154,2009). TRAIL activates the extrinsic apoptotic pathway by binding to thecell membrane receptors death receptor 4 (DR4, also called TRAIL-R1)and/or death receptor 5 (DR5, also called TRAIL-R2 or Apo2), thusinducing killing of susceptible cells. More specifically, the active,soluble form of TRAIL is a self-assembling, non-covalent homotrimer thatbinds the extracellular domain of three receptor molecules with highaffinity. This induces oligomerization of the intracellular deathdomains and formation of homomeric or heteromeric complexes (FC Kischkelet al., Immunity 12:611-620, 2000), followed by recruitment ofFas-associated protein with death domain (FADD) and formation of thedeath inducing signaling complex (DISC) leading to activation ofcaspases and then apoptosis, programmed cell death (see van Roosmalen etal., op. cit.).

Many cancer cells express DR4 and/or DR5, and TRAIL is able toselectively induce apoptosis of cancer cells (reviewed in J Lemke etal., Cell Death Differ 21: 1350-1364, 2014) and in cross-linked form oftumor endothelial cells (NS Wilson et al., Cancer Cell 22:80-90, 2012).TRAIL itself and various TRAIL-receptor agonists have demonstratedstrong antitumor activity against cancer cell lines and in preclinicalmodels (e.g., A Ashkenazi et al., J Clin Invest 104:155-162, 1999).These include recombinant human TRAIL (rhTRAIL; dulanermin) consistingof the extracellular region of human TRAIL (R Pitti et al., J. Biol Chem271: 12687-12690, 1996), and a number of agonist monoclonal antibodies(mAbs) against DR4 or DR5, including murine, chimeric, humanized andhuman mAbs (see van Roosmalen et al., op. cit.). Such mAbs include the4H6.17.8 mAb (designated herein 4H6; U.S. Pat. No. 7,252,994) andmapatumumab (HGS-ETR1; Pukac et al., Br J Cancer 92:1430-1441, 2005)against DR4; and the 3H3.14.5 mAb (designated herein 3H3; U.S. Pat. No.6,252,050), conatumumab (AMG 655; P Kaplan-Lefko et al., Cancer BiolTher 9:618-631, 2010), drozitumab (Apomab; C Adams et al., Cell DeathDiffer 15:751-761, 2008), lexatumumab (HGS-ETR2; G Georgakis et al., BrJ Haematol 130:501-510, 2005) and TRA-8 and its humanized formtigatuzumab (CS-1008, A Yada et al., Ann Oncol 19:1060-1067, 2008)against DR5; as well as other mAbs listed in van Roosmalen, op. cit.(see Table 1 on p. 450).

However, while all these agents are very effective at killing tumorcells in vitro and generally in animal models (see references citedabove), none of them has demonstrated strong activity in clinicaltrials, whether used alone or in combination with other agents, and nonehas advanced into Phase III (reviewed in PM Holland, Cytokine GrowthFactor Rev 25:185-193, 2014, and in Lemke et al., op cit. See especiallyTable 1 in Holland and Tables 2 and 3 in Lemke et al., and referencescited therein). For the mAbs, one reason may be the requirement forcross-linking of the mAbs to oligomerize the death receptors to triggerthe apoptosis pathway. Such a requirement could be satisfied in vivo bybinding of the Fc domain of the mAbs to Fc gamma receptors (FcγR) onimmune cells, but there may be too few immune cells infiltrating thetumors or such binding may not be of high enough affinity. Cancer cellsmay also express inhibitors of apoptosis proteins (see Lemke et al., op.cit.). To improve efficacy, modified forms of TRAIL are being developed,including fusions with other protein domains to enhance stability,oligomerization and/or targeting (Lemke et al., op cit. and Holland, op.cit.). Similarly, constructs comprising multiple antibody bindingdomains to DR4 and/or DR5 have been developed (K Miller et al., JImmunol 170:4854-4861, 2003; W Wang et al., Immunol Cell Biol91:360-367, 2013; J E Allen et al., Mol Cancer Ther 11:2087-95, 2012; HHuet et al., Cancer Res 72 abstract 3853, 2012; WO 2014/022592; WO2015/017822) but have either not entered or not been successful inclinical trials (KP Papadopoulos et al., Cancer Chemother Pharmacol;Feb. 27, 2015, PMID: 25721064); their very unnatural structure may limittheir clinical utility. In another approach, combination orco-administration of TRAIL and the anti-DR5 mAb AMG 655 was moreeffective than either agent alone in vitro and in vivo (J D Graves etal., Cancer Cell 26:177-189, 2014).

SUMMARY OF THE CLAIMED INVENTION

The invention provides monoclonal antibodies (mAbs) that bind to DR4 andDR5, such as D114 and G4.2 and their humanized forms. In one embodiment,the invention provides a bispecific monoclonal antibody comprising twopairs of a heavy chain and a light chain, wherein each heavy/light chainpair comprises a domain that binds to DR4 and a domain that binds toDR5. In other embodiments, the invention provides a multimericmonoclonal antibody that has four binding domains for DR4, or fourbinding domains for DR5. In preferred embodiments, the antibody has theform of a Bs(scFv)₄-IgG antibody as this term is defined below. In anyof the bispecific or multimeric mAbs, each binding domain is preferablyfrom a humanized or human mAb, such as humanized forms of D114 and G4.2.Also, any mAb of the invention can contain a constant region comprisingmutations that enhance binding to a cellular Fc gamma receptor (FcγR),for example FcγRIIb, e.g., the S267E and/or L328F mutations in a gamma-1constant region (according to Kabat numbering with EU index), preferablytwo or more such mutations. Advantageously, the mAb inhibits growth of ahuman tumor xenograft in a mouse. In another aspect, a pharmaceuticalcomposition comprising any of these mAbs is provided. In a third aspect,such a pharmaceutical composition is administered to a patient to treatcancer or other disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, B. Schematic diagrams of the Bs(scFv)₄-IgG antibody formatshowing individual variable and constant regions (A) or the domainsformed by folding together of each light chain region with respectiveheavy chain region (B). V_(H)1 (respectively V_(L)1)=heavy (resp. light)chain variable region of first antibody; and similarly for V_(H)2 andV_(L)2 of second antibody. C_(H)1, C_(H)2, C_(H)3 (resp. C_(L))=heavy(resp. light) constant region regions; H=hinge region. V1 (resp.V2)=full variable domain of first (resp. second) antibody.

FIGS. 2A-D. (A) Graph showing binding of 4H6 and D114 mAbs to DR4. (B)Graph showing inhibition of binding of Apo2L to DR4 by 4H6 and D114mAbs. (C) Graph showing binding of 3H3 and G4.2 mAbs to DR5. (D) Graphshowing inhibition of binding of Apo2L to DR5 by 3H3 and G4.2 mAbs. mIgGis mouse negative control mAb.

FIGS. 3A-D. Cell viability of H60 lung tumor cells (A) and SW480 colontumor cells (B) after treatment with 4H6 and D114 mAbs in the presenceof goat anti-mouse IgG-Fc antibody (anti-mIgG-Fc); cell viability of H60lung tumor cells (C) and COLO 205 colon tumor cells (D) after treatmentwith 3H3 and G4.2 mAbs in the presence of anti-mIgG-Fc.

FIGS. 4A-D. Amino acid sequences of the mature heavy (A) and light (B)chain variable regions of the 4H6 mAb, and of the mature heavy (C) andlight (D) chain variable regions of the 3H3 mAb, using the 1-letter code(SEQ ID NOS:1-4 respectively).

FIGS. 5A-D. Amino acid sequences of the mature variable regions of theHuD114-L1 light chain (A) and HuD114-H1 and HuD114-H2 heavy chains (B)are shown aligned with mouse D114 and human acceptor V regions; andamino acid sequences of the mature variable regions of the HuG4.2-L1 andHuG4.2-L2 light chains (C) and HuG4.2-H1 and HuG4.2-H2 heavy chains (D)are shown aligned with mouse G4.2 and human acceptor V regions. Thesequences are designated (A) SEQ ID NOS. 5-7, (B) SEQ ID NOS. 8-11, (C)SEQ ID NOS. 12-15, and (D) SEQ ID NOS. 16-19. The CDRs are underlined inthe D114 (respectively G4.2) sequences. CDR-L1, -L2, -L3, -H1, -H2 and-H3 of D114 are designated SEQ ID NOS. 20-25 respectively and of G4.2are designated SEQ ID NOS. 26-31) and the amino acids substituted withmouse D114 (resp. G4.2) amino acids are double underlined in the HuD114(resp. HuG4.2) sequences. The 1-letter amino acid code and Kabatnumbering system are used for both the light and heavy chain in allfigures herein.

FIGS. 6A, B. Amino acid sequences (1-letter code) of the complete heavy(A) and light (B) chains of the B-3H3/4H6-hFc** bispecific antibody (SEQID NOS. 32 and 33), including signal peptides that are cleaved off andthus not present in the mature proteins, which mature proteins aredesignated SEQ ID NOS: 34 and 35. Arrows under the sequences separate inorder the signal peptide (shown struck through), 4H6 V_(H) region (A) orrespectively 3H3 V_(H) region (B), linker sequence (shown underlined),4H6 V_(L) region (A) or respectively 3H3 V_(L) region (B), and heavychain (A) or light chain (B) constant region. The mutated amino acids267E and 328F are double underlined.

FIGS. 7A, B. Cell viability of SW480 colon tumor cells after treatmentby the indicated agents in the absence (A) and presence (B) of goatanti-hIgG-Fc, as measured by a WST-8 assay. hIgG is control human mAb inthe figures herein.

FIGS. 8A, B. Cell viability of H460 lung tumor cells after treatment bythe indicated agents without goat anti-hIgG-Fc (A) and in the presenceof human PBMCs (B).

FIGS. 9A, B. (A) Cell viability of COLO 205 colon tumor cells afterincubation with 0.5 μg/mL of the indicated agents with PBMCs from 4human donors. (B) Cell viability of the indicated cells after incubationwith 1.0 μg/mL of the indicated agents with PBMCs. Error bars arestandard error of the mean (SEM) in the figures herein.

FIGS. 10A, B. (A) Cell viability of COLO 205 cells after treatment bythe indicated agents in the presence of PBMCs (short dashes, 4H6-hFc;long dashes, 4H6-hFc*). (B) Inhibition of growth of COLO 205 colon tumorxenografts by the indicated agents.

FIGS. 11A-C. Inhibition of growth of COLO 205 colon tumor xenografts (A,C) and SW480 colon tumor xenografts (B) by the indicated agents

FIGS. 12A, B. Inhibition of growth of COLO 205 colon tumor xenografts(A) and MIA PaCa-2 pancreatic tumor xenografts (B) by the indicatedagents.

FIGS. 13A, B. Graphs showing binding of the indicated anti-DR4 mAbs toDR4 (A) and anti-DR5 mAbs to DR5 (B). In all figure legends herein, the-Fc and -Fc** suffixes mean the same as -hFc and -hFc** respectively(e.g., HuD114-Fc #1 means the same as HuD114-hFc #1, etc.) In this andthe following figures, as indicated by the legends, generally solidlines denote the hFc** form of mAbs, dashed lines denote the hFc or hFc*form, and dotted lines denote mouse mAbs.

FIGS. 14A, B. Cell viability of COLO 205 colon tumor cells aftertreatment by anti-DR4 mAbs (A) and anti-DR5 mAbs (B) in the presence ofgoat anti-hIgG-Fc. Different concentration units are used in (A) and(B).

FIGS. 15A-D. Cell viability of COLO 205 colon tumor cells (A), SW480colon tumor cells (B), COLO 205 cells (C) and MIA PaCa-2 pancreatictumor cells (D) after treatment by the indicated mAbs, in the presenceof anti-mIgG-Fc in the case of the mouse TRA-8 mAb or goat anti-hIgG-Fcin the case of humanized or human mAbs.

FIGS. 16A-B. Cell viability of H60 lung tumor cells (A) and SW480 colontumor cells (B) after treatment by the indicated agents in the presenceof goat anti-hIgG-Fc.

FIGS. 17A-F. Cell viability of COLO 205 colon tumor cells (A, C and E)and H60 lung tumor cells (B, D, and F) after treatment by the indicatedagents in the presence of human PBMCs.

FIGS. 18A-D. Inhibition of growth of COLO 205 colon tumor xenografts(A), H60 lung tumor xenografts (B), COLO 205 xenografts (C), and Ramoslymphoma xenografts (D) by the indicated agents.

FIGS. 19A, B. Inhibition of growth of COLO 205 colon tumor xenografts(A) and MIA PaCa-2 pancreatic tumor xenografts (B) by the indicatedagents.

FIGS. 20A-D. (A, C) Results of ELISA assay measuring simultaneousbinding of each indicated agent to DR4 and DR5. In (A), the curves for4H6-hFc** and 3H3-hFc** superimpose and cannot be distinguished. (B, D)Cell viability of COLO 205 cells after treatment by the indicated agentsin the absence of goat anti-mIgG-Fc.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Antibodies

As used herein, “antibody” means a protein containing one or moredomains capable of binding an antigen, where such domain(s) are derivedfrom or homologous to the variable domain of a natural antibody. Amonoclonal antibody (“mAb”) is simply a unique species of antibody, incontrast to a mixture of different antibodies. The antibodies describedherein are generally monoclonal, unless otherwise indicated by thecontext. An “antigen” of an antibody means a compound to which theantibody specifically binds and is typically a polypeptide, but can alsobe a small peptide or small-molecule hapten or carbohydrate or othermoiety. Examples of antibodies include natural, full-length tetramericantibodies; antibody fragments such as Fv, Fab, Fab′ and (Fab′)₂;single-chain (scFv) antibodies (Huston et al., Proc. Natl. Acad. Sci.USA 85:5879, 1988; Bird et al., Science 242:423, 1988); single-armantibodies (Nguyen et al., Cancer Gene Ther 10:840, 2003); andbispecific, chimeric and humanized antibodies, as these terms arefurther explained below. Antibodies may be derived from any vertebratespecies, including chickens, rodents (e.g., mice, rats and hamsters),rabbits, camels, primates and humans. An antibody comprising a constantdomain may be of any of the known isotypes IgG, IgA, IgM, IgD and IgEand their subtypes, e.g., human IgG1, IgG2, IgG3, IgG4 and mouse IgG1,IgG2a, IgG2b, and IgG3, and their allotypes and isoallotypes, includingpermutations of residues occupying polymorphic positions in allotypesand isoallotypes. An antibody can also be of chimeric isotype, that is,one or more of its constant (C) regions can contain regions fromdifferent isotypes, e.g., a gamma-1 C_(H)1 region together with hinge,C_(H)2 and/or C_(H)3 domains from the gamma-2, gamma-3 and/or gamma-4genes. The antibody may also contain replacements in the constantregions to reduce or increase effector function such ascomplement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S.Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar etal., Proc Natl Acad Sci USA 103:4005, 2006), or to prolong half-life inhumans (see, e.g., Hinton et al., J Biol Chem 279:6213, 2004).

A natural antibody molecule is generally a tetramer consisting of twoidentical heterodimers, each of which comprises one light chain pairedwith one heavy chain. Each light chain and heavy chain consists of avariable (V_(L) for the light chain, or V_(H) for the heavy chain, or Vfor both) region followed by a constant (C_(L) or C_(H), or C) region.The C_(H) region itself comprises C_(H)1, hinge (H), C_(H)2, and C_(H)3regions. In 3-dimensional (3D) space, the V_(L) and V_(H) regions foldup together to form a V domain, which is also known as a binding domainsince it binds to the antigen. The C_(L) region folds up together withthe C_(H)1 region, so that the light chain V_(L)-C_(L) and theV_(H)-C_(H)1 region of the heavy chain together form a part of theantibody known as a Fab: a naturally “Y-shaped” antibody thus containstwo Fabs, one from each heterodimer, forming the arms of the Y. TheC_(H)2 region of one heterodimer is positioned opposite the C_(H)2region of the other heterodimer, and the respective C_(H)3 regions foldup with each other, forming together the single Fc domain of theantibody (the base of the Y), which interacts with other components ofthe immune system.

Within each light or heavy chain variable region, there are three shortsegments (averaging about 10 amino acids in length) called thecomplementarity determining regions (“CDRs”). The six CDRs in anantibody variable domain (three from the light chain and three from theheavy chain) fold up together in 3D space to form the actual antibodybinding site which locks onto the target antigen. The position andlength of the CDRs have been precisely defined by E Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1983, 1987, 1991. The part of a variableregion not contained in the CDRs is called the framework, which formsthe environment for the CDRs. Chothia et al., J. Mol. Biol. 196:901,1987, have defined the related concept of hypervariable regions or loopsdetermined by structure.

As used herein, a “genetically engineered” mAb is one for which thegenes have been constructed or put in an unnatural environment (e.g.,human genes in a mouse or on a bacteriophage) with the help ofrecombinant DNA techniques, and therefore includes chimeric antibodiesand humanized antibodies, as described below, but would not encompass amouse or other rodent mAb made with conventional hybridoma technology. Achimeric antibody (or respectively chimeric antibody light or heavychain) is an antibody (or respectively antibody light or heavy chain) inwhich the variable region of a mouse (or other non-human species)antibody (or respectively antibody light or heavy chain) is combinedwith the constant region of a human antibody; their construction bymeans of genetic engineering is well-known. Such antibodies retain thebinding specificity of the mouse antibody, while being about two-thirdshuman.

A humanized antibody is a genetically engineered antibody in which CDRsfrom a non-human “donor” antibody (e.g., chicken, mouse, rat, rabbit orhamster) are grafted into human “acceptor” antibody sequences, so thatthe humanized antibody retains the binding specificity of the donorantibody (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089;Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No. 6,407,213; Adair,U.S. Pat. Nos. 5,859,205 6,881,557; Foote, U.S. Pat. No. 6,881,557). Theacceptor antibody sequences can be, for example, a mature human antibodysequence, a consensus sequence of human antibody sequences, a germlinehuman antibody sequence, or a composite of two or more such sequences.Thus, a humanized antibody is an antibody having some or all CDRsentirely or substantially from a donor antibody and variable regionframework sequences and constant regions, if present, entirely orsubstantially from human antibody sequences. Similarly, a humanizedlight chain (respectively heavy chain) has at least one, two and usuallyall three CDRs entirely or substantially from a donor antibody light(resp. heavy) chain, and a light (resp. heavy) chain variable regionframework and light (resp. heavy) chain constant region, if present,substantially from a human light (resp. heavy) acceptor chain. Ahumanized antibody generally comprises a humanized heavy chain and ahumanized light chain. A CDR in a humanized antibody is substantiallyfrom a corresponding CDR in a non-human antibody when at least 85%, 90%,95% or 100% of corresponding amino acids (as defined by Kabat) areidentical between the respective CDRs. The variable region framework orconstant region of an antibody chain are substantially from a humanvariable region or human constant region respectively when at least 85,90, 95 or 100% of corresponding amino acids (as defined by Kabat) areidentical.

Here, as elsewhere in this application, percentage sequence identitiesare determined with antibody sequences maximally aligned by the Kabatnumbering convention (Eu index for the C_(H) region). After alignment,if a subject antibody region (e.g., the entire mature variable region ofa heavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

In order to retain high binding affinity in a humanized antibody, atleast one of two additional structural elements can be employed. See,U.S. Pat. Nos. 5,530,101 and 5,585,089, incorporated herein byreference, which provide detailed instructions for construction ofhumanized antibodies. In the first structural element, the framework ofthe heavy chain variable region of the acceptor or humanized antibody ischosen to have high sequence identity (between 65% and 95%) with theframework of the heavy chain variable region of the donor antibody, bysuitably selecting the acceptor antibody heavy chain from among the manyknown human antibodies. In the second structural element, inconstructing the humanized antibody, selected amino acids in theframework of the human acceptor antibody (outside the CDRs) are replacedwith corresponding amino acids from the donor antibody, in accordancewith specified rules. Specifically, the amino acids to be replaced inthe framework are chosen on the basis of their ability to interact withthe CDRs. For example, the replaced amino acids can be adjacent to a CDRin the donor antibody sequence or within 4-6 angstroms of a CDR in thehumanized antibody as measured in 3-dimensional space.

Other approaches to design humanized antibodies may also be used toachieve the same result as the methods in U.S. Pat. Nos. 5,530,101 and5,585,089 described above, for example, “superhumanization” (see Tan etal. J Immunol 169: 1119, 2002, and U.S. Pat. No. 6,881,557) or themethod of Studnicak et al., Protein Eng. 7:805, 1994. Moreover, otherapproaches to produce genetically engineered, reduced-immunogenicitymAbs include “reshaping”, “hyperchimerization” andveneering/resurfacing, as described, e.g., in Vaswami et al., Annals ofAllergy, Asthma and Immunology 81:105, 1998; Roguska et al. Protein Eng.9:895, 1996; and U.S. Pat. Nos. 6,072,035 and 5,639,641. Veneeredantibodies are made more human-like by replacing specific amino acids inthe variable region frameworks of the non-human donor antibody that maycontribute to B- or T-cell epitopes, for example exposed residues(Padlan, Mol. Immunol. 28:489, 1991). Other types of geneticallyengineered antibodies include human antibodies made using phage displaymethods (Dower et al., WO91/17271; McCafferty et al., WO92/001047;Winter, WO92/20791; and Winter, FEBS Lett. 23:92, 1998, each of which isincorporated herein by reference) or by using transgenic animals(Lonberg et al., WO93/12227; Kucherlapati WO91/10741, each of which isincorporated herein by reference).

The terms “antibody” or “mAb” also encompass bispecific antibodies. A“bispecific antibody” is an antibody that contains a first domainbinding to a first antigen and a second (different) domain binding to asecond antigen, where the first and second domains are derived from orhomologous to variable domains of natural antibodies. The first antigenand second antigen may be the same antigen, in which case the first andsecond domains can bind to different epitopes on the antigen. The termbispecific antibody encompasses multispecific antibodies, which inaddition to the first and second domains contain one or more otherdomains binding to other antigens and derived from or homologous tovariable domains of natural antibodies. The term bispecific antibodyalso encompasses an antibody containing a first binding domain derivedfrom or homologous to a variable domain of a natural antibody, and asecond binding domain derived from another type of protein, e.g., theextracellular domain of a receptor, (a “bispecificantibody-immunoadhesin”). And the term bispecific antibody furtherencompasses a “two-in-one” antibody with a dual specificity bindingdomain (G Schaefer et al., Cancer Cell 20:472-486, 2011).

Bispecific antibodies have been produced in a variety of forms (see,e.g., Kontermann, MAbs 4:182-197, 2012 and references cited therein),for example single chain variable fragment (scFv), Fab-scFv, andscFv-scFv fusion proteins (Coloma et al., Nat Biotechnol 15:125-126,1997; Lu et al., J Immunol Methods 267:213-226, 2002; Mallender, J BiolChem 269:199-206, 1994), Bs(scFv)₄-IgG (Z Zuo et al., Protein Eng 13:361-367, 2000), double variable domain antibodies (C Wu et al., NatBiotechnol 25:1290-1297, 2007), and diabodies (Holliger et al., ProcNatl Acad Sci USA 90:6444-6448, 1993). Bispecific F(ab′)₂ antibodyfragments have been produced by chemical coupling (Brennan et al.,Science 229:81, 1985) or by using leucine zippers (Kostelny et al., JImmunol 148:1547-1553, 1992). A more naturally shaped bispecificantibody, with each heavy chain-light chain pair having a different Vregion, can be made, e.g., by chemically cross-linking the two heavychain-light chain pairs produced separately (Karpovsky et al., J Exp Med160:1686-1701, 1984), Naturally shaped bispecific antibodies can also beproduced by expressing both required heavy chains and light chains in asingle cell, made by fusing two hybridoma cell lines (a “quadroma”;Milstein et al., Nature 305: 537-540) or by transfection. Association ofthe correct light and heavy chains expressed in a cell to form thedesired bispecific antibody can be promoted by using “knobs-into-holes”technology (Ridgway et al., Protein Eng 9:617-621, 1996; Atwell et al.,J Mol Biol 270:26-35, 1997; and U.S. Pat. No. 7,695,936); optionallywith exchange or “crossing over” of heavy chain and light chain domainswithin the antigen binding fragment (Fab) of one light chain—heavy chainpair, thus creating bispecific antibodies called “CrossMabs” (Schaeferet al., Proc Natl Acad Sci USA 108:11187-11192, 2011; WO 2009/080251; WO2009/080252; WO 2009/080253).

Related to the concept of bispecific antibodies are “multimeric”antibodies, which are mAbs that contain more than two binding domains,each binding to the same antigen. Many of the formats for bispecificmAbs, for example Bs(scFv)₄-IgG and double variable domain, may beadapted to make multimeric mAbs by using the same variable domain aseach binding domain. So for example, a multimeric Bs(scFv)₄-IgG antibodywould contain 4 copies of the same binding domain.

An antibody is said to bind “specifically” to an antigen if it binds toa significantly greater extent than irrelevant antibodies not bindingthe antigen, and thus typically has binding affinity (K_(a)) of at leastabout 10⁶ but preferably 10⁷, 10⁸, 10⁹ or 10¹⁰ M⁻¹ for the antigen.Generally, when an antibody is said to bind to an antigen, specificbinding is meant. If an antibody is said not to bind an antigen, it ismeant that any signal indicative of binding is not distinguishablewithin experimental error from the signal of irrelevant controlantibodies. The epitope of a mAb is the region of its antigen to whichthe mAb binds. Two antibodies are judged to bind to the same oroverlapping epitopes if each competitively inhibits (blocks) binding ofthe other to the antigen. Competitively inhibits binding means that a 1×or 5× excess of one antibody inhibits binding of the other by at least50% but preferably 75%, or that a 10×, 20× or 100× excess of oneantibody inhibits binding of the other by at least 75% but preferably90% or even 95% or 99% as measured in a competitive binding assay (see,e.g., Junghans et al., Cancer Res. 50:1495, 1990). One mAb (the secondmAb) is said to “fully” compete for binding an antigen with another mAb(the first mAb) if the inhibitory concentration 50 (IC50) of the secondmAb to inhibit binding (of the first mAb) is comparable to, that is,within 2-fold or 3-fold, of the IC50 of the first mAb to inhibit bindingof itself, in competitive binding assays. A second mAb is said to“partially” compete for binding an antigen with a first mAb if the IC50of the second mAb to inhibit binding (of the first mAb) is substantiallygreater than, e.g., greater than 3-fold or 5-fold or 10-fold, the IC50of the first mAb to inhibit binding. In general, two mAbs have the sameepitope on an antigen if each fully competes for binding to the antigenwith the other, and have overlapping epitopes if at least one mAbpartially competes for binding with the other mAb. Alternatively, twoantibodies have the same epitope if essentially all amino acid mutationsin the antigen that reduce or eliminate binding of one antibody reduceor eliminate binding of the other, while two antibodies have overlappingepitopes if some amino acid mutations that reduce or eliminate bindingof one antibody reduce or eliminate binding of the other.

2. Anti-DR4 and Anti-DR5 Antibodies

A monoclonal antibody that binds DR4, (i.e., an anti-DR4 mAb), orrespectively an antibody that binds DR5 (i.e., an anti-DR5 mAb) is saidto be agonist if binding of the mAb to the cell membrane receptor DR4 orrespectively DR5 transmits an apoptotic signal to at least some types ofcells, thus inducing apoptosis. Such an antibody may be blocking ornon-blocking, i.e., inhibit or not inhibit binding of Apo2L/TRAIL to DR4or DR5 respectively. An agonist mAb of the invention, which may be abispecific antibody binding to either DR4 and DR5 and a second antigen,or to both DR4 and DR5, at a concentration of, e.g., 0.1, 1, 10, 100,1000, or 10,000 ng/mL, inhibits cell viability or induces apoptosis byapproximately 25%, 50%, 75%, 90%, 95%, 99% or more, as measured forexample by inhibition of cellular metabolism, e.g., using the WST-8assay. Such cell line may for example be the COLO 205 or SW480 colontumor lines, H460 lung cancer line, or BxPC-3 pancreatic cancer cellline. Such activity may be obtained by use of the mAb alone, or in thepresence of either human cells such as peripheral blood mononuclearcells or of an antibody that cross-links the mAb, for example goatanti-IgG-Fc (at e.g., 10 μg/mL). The activity is typically measuredafter incubation of the mAb plus cells plus any other agents overnightat 37° C.

MAbs to be used in the present invention are preferably specific for DR4or respectively DR5, that is they do not (specifically) bind, or onlybind to a much lesser extent (e.g., less than ten-fold), proteins thatare related to DR4 or DR5 such as the tumor necrosis factor (TNF)receptors TNFR1 and TNFR2 and other members of the TNFR superfamily,other death receptors such as Apo-1 (Fas), and the decoy receptors DcR1and DcR2. MAbs to be used in the invention typically have a bindingaffinity (K_(a)) for DR4 or DR5 of at least 10⁷ M⁻¹ but preferably 10⁸M⁻¹ or higher, and most preferably 10⁹ M⁻¹, 10¹⁰ M⁻¹ or 10¹¹ M⁻¹ orhigher. Such a mAb binds human DR4 or DR5, but advantageously also DR4or DR5 from other species, e.g., mice or non-human primates such ascynomolgus monkeys, ideally with binding affinity similar to (e.g.,within 10-fold) the binding affinity to human DR4 or DR5. The sequenceof human DR4 and DR5 are respectively provided, e.g., by G Pan et al.,Science. 276:111-113, 1997 (GenBank: AAC51226.1) and H Walczak et al.,EMBO J 16:5386-5397, 1997 (GenBank: CAG46696.1).

Exemplary antibodies of the invention are D114 and G4.2 and theirchimeric and humanized forms such as HuD114 and HuG4.2. These as well asother anti-DR4 mAbs such as 4H6 (produced by the hybridoma ATCCHB-12455; see U.S. Pat. No. 7,252,994), and anti-DR5 mAbs such as 3H3(produced by the hybridoma ATCC HB-12534; see U.S. Pat. No. 6,252,050)or other antibody includes all the CDRs of 4H6 or 3H3 can be used in thebispecific and multimeric mAbs of the invention, with chimeric,humanized or human agonist mAbs preferred for such use. Otherembodiments of the invention include a preferably agonist anti-DR4 oranti-DR5 mAb—either an ordinary IgG or a bispecific or multimericantibody—comprising mutations in the constant region that increasebinding to an Fc receptor such as a receptor for IgG antibodies (FcγR),e.g., the FcγRIIb receptor, for example the HuD114-hFc** andHuG4.2-hFc** mAbs described below. Exemplary mutations are the singleG236D, L328F, S239D, and S267E mutations, and preferably doublemutations such as G236D/S267E, S239D/S267E and most preferablyS267E/L328F that provide greater binding affinity to an FcγR than eitherconstituent single mutation (SY Chu et al., Mol Immunol 45:3926-3933,2008), as well as other mutations at these amino acid positions, usingthe Kabat numbering system, which is used to define all amino acidpositions set forth herein. Of these, the S267E single mutation has beenshown to increase the potency of an anti-DR5 mAb in vitro and in vivo (FLi et al., Proc Natl Acad Sci USA 109:10966-10971, 2012). Mostpreferably, the mAb of the invention inhibits growth of a human tumorxenograft in a mouse as assessed by any of the assays in the Examples orotherwise known in the art.

MAbs that have CDRs (for example three CDRs in the light chain and threeCDRs in the heavy chain), as defined by Kabat, that individually orcollectively are at least 90%, 95% or 98% or completely identical to theCDRs of D114 (respectively G4.2) in amino acid sequence and thatmaintain its functional properties, e.g., a humanized D114 (resp. G4.2)mAb, or which differ from D114 (resp. G4.2) by a small number offunctionally inconsequential amino acid substitutions (e.g.,conservative substitutions, as defined below), deletions, or insertionsare also an embodiment of the invention, with or without the mutationsdescribed above.

Once a single, archetypal anti-human-DR4 (respectively anti-human-DR5)mAb, for example D114 (resp. G4.2), has been isolated that has thedesired properties described herein, it is straightforward to generateother mAbs with similar properties by using art-known methods, includingmAbs that compete with D114 (resp. G4.2) for binding to DR4 (resp. DR5)and/or have the same epitope. For example, mice may be immunized withthe extracellular domain of DR4 (resp. DR5), hybridomas produced, andthe resulting mAbs screened for the ability to compete with D114 (resp.G4.2) for binding to DR4 (resp. DR5). Mice can also be immunized with asmaller fragment of DR4 (resp. DR5) containing the epitope to which D114(resp. G4.2) binds. The epitope can be localized by, e.g., screening forbinding to a series of overlapping peptides spanning DR4 (resp. DR5).Mouse mAbs generated in these ways can then be humanized. Alternatively,the method of Jespers et al., Biotechnology 12:899, 1994, which isincorporated herein by reference, may be used to guide the selection ofmAbs having the same epitope and therefore similar properties to D114(resp. G4.2). Using phage display, first the heavy chain of D114 (resp.G4.2) is paired with a repertoire of (preferably human) light chains toselect a DR4-binding (resp. DR5-binding) mAb, and then the new lightchain is paired with a repertoire of (preferably human) heavy chains toselect a (preferably human) DR4-binding (resp. DR5-binding) mAb havingthe same epitope as D114 (resp. G4.2). Alternatively variants of D114(resp. G4.2) can be obtained by mutagenesis of DNA encoding the heavyand light chains of D114 (resp. G4.2).

Genetically engineered mAbs, e.g., chimeric or humanized or bispecificmAbs, may be expressed by a variety of art-known methods. For example,genes encoding their light and heavy chain V regions may be synthesizedfrom overlapping oligonucleotides and inserted together with available Cregions into expression vectors (e.g., commercially available fromInvitrogen) that provide the necessary regulatory regions, e.g.,promoters, enhancers, poly A sites, etc. Use of the CMVpromoter-enhancer is preferred. The expression vectors may then betransfected using various well-known methods such as lipofection orelectroporation into a variety of mammalian cell lines such as CHO ornon-producing myelomas including Sp2/0 and NSO, and cells expressing theantibodies selected by appropriate antibiotic selection. See, e.g., U.S.Pat. No. 5,530,101. Larger amounts of antibody may be produced bygrowing the cells in commercially available bioreactors. Hence, theinvention provides cell lines expressing any of the mAbs describedherein.

Once expressed, the mAbs of the invention including bispecific mAbs maybe purified according to standard procedures of the art such asmicrofiltration, ultrafiltration, protein A or G affinitychromatography, size exclusion chromatography, anion exchangechromatography, cation exchange chromatography and/or other forms ofaffinity chromatography based on organic dyes or the like. Substantiallypure antibodies of at least about 90 or 95% homogeneity are preferred,and 98% or 99% or more homogeneity most preferred, for pharmaceuticaluses. It is also understood that when the mAb is manufactured byconventional procedures, one to several amino acids at the amino orcarboxy terminus of the light and/or heavy chain, such as the C-terminallysine of the heavy chain, may be missing or derivatized in a proportionor all of the molecules, and such a composition is still considered tobe the same mAb.

3. Bispecific and Multimeric Antibodies

In one embodiment, the invention provides a bispecific monoclonalantibody comprising at least one binding domain that binds to DR4 and atleast one binding domain that binds to DR5. Such an antibody is called abispecific DR4/DR5 antibody or mAb herein. In preferred embodiments,each binding domain is from a humanized or human mAb. In preferredembodiments, the bispecific antibody comprises two or more bindingdomains that bind to DR4 and two or more binding domains that bind toDR5; often the two binding domains to DR4 are the same and the twobinding domains to DR5 are the same. In any case, the bispecific mAb ispreferably agonist as described above, i.e., induces an apoptotic signalthrough DR4 and/or DR5 in susceptible cells such as cancer cells.Exemplary bispecific mAbs comprise the binding domain of the 4H6anti-DR4 mAb or the D114 mAb disclosed herein or their humanized forms,and the binding domain of the 3H3 anti-DR5 mAb or the G4.2 mAb disclosedherein or their humanized forms.

The bispecific antibody of the invention may be in any format, such asany of those listed in Kontermann, op. cit. In one preferred embodiment,the bispecific antibody is in the Bs(scFv)4-IgG format described in Zuoet al., op. cit. and illustrated in FIGS. 1A, B. In this format, onebinding domain in single chain (scFv) form is connected to the C_(L)region and thus becomes the N-terminal domain of the light chain, whilethe other binding domain in scFv form is connected to the C_(H)1 domainand thus becomes the N-terminal domain of the heavy chain; two lightchains and two heavy chains form a homodimer as in an ordinary IgGantibody, but containing two of each binding domain. Thus, an advantageof the Bs(scFv)4-IgG format is that it is a homodimer, with the sameheavy chain and light chain in each monomer, so that no precautions needto be taken to ensure correct heterodimerization. The linker within eachscFv connecting the V_(L) and V_(H) regions is often chosen as (G₄S)₃GS(SEQ ID NO:36) or ASGS(G₄S)₃ (SEQ ID NO:37). Each scFv binding domainmay be in the form V_(L)-linker-V_(H) or in the form V_(H)-linker-V_(L)(as shown in FIG. 1A), and either binding domain may be part of thelight chain while the other is part of the heavy chain, so in total2×2×2=8 variants of a Bs(scFv)4-IgG antibody can be made from two givenbinding domains (e.g., those of HuD114 and HuG4.2), which may havediffering properties.

In another embodiment of the invention, the bispecific antibody is inthe double variable domain format described in, e.g., Wu et al., op.cit., (see FIG. 1A with labeling therein). Like Bs(scFv)4-IgG mAbs, sucha bispecific mAb is a homodimer, with each monomer containing one ofeach of the binding domains, linked in the sequence V1V2-constantdomain. A variety of peptide linkers may be used to connect the firstand second variable regions, e.g., ASTKGPSVFPLAP (SEQ ID NO:38) in theheavy chain and RTVAAPSVIFIPP (SEQ ID NO:39) in the light chain, orASGS(G₄S)₃ (SEQ ID NO:37) in both chains. For example, in such abispecific mAb, the variable domain of HuG4.2 could be the first domain,the variable domain of HuD114 could be the second domain; the linkerscould be the former ones mentioned above, and the constant region couldbe of the human IgG1, kappa isotype.

In another embodiment of the invention, the bispecific antibody is inthe double variable domain format described in, e.g., Wu et al., op.cit., (see FIG. 1A with labeling therein). Like Bs(scFv)4-IgG mAbs, sucha bispecific mAb is a homodimer, with each monomer containing one ofeach of the binding domains, linked in the sequence V1-V2-constantdomain. A variety of peptide linkers may be used to connect the firstand second variable regions, e.g., ASTKGPSVFPLAP in the heavy chain andRTVAAPSVIFIPP in the light chain, or ASGS(G₄S)₃ in both chains. Forexample, in such a bispecific mAb, the variable domain of HuG4.2 couldbe the first domain, the variable domain of HuD114 could be the seconddomain; the linkers could be the former ones mentioned above, and theconstant region could be of the human IgG1, kappa isotype.

In other preferred embodiments of the invention, one monomer of theanti-DR4 mAb comprising a light and heavy chain pairs with one monomerof the anti-DR5 mAb comprising a light and heavy chain to form aheterodimer with the normal configuration of an IgG molecule. If allfour chains are to be expressed in a cell, formation of the desiredheterodimer bispecific antibodies instead of homodimers is promoted byinserting knobs and holes into the C_(H)3 regions of the respectiveheavy chains (Ridgway et al., Protein Eng 9:617-21, 1996; Atwell et al.,J Mol Biol 270:26-35, 1997; and U.S. Pat. No. 7,695,936), while correctpairing of the light and heavy chains to form each anti-DR4 and anti-DR5monomer is promoted by “crossing over” of heavy chain and light chaindomains within one of the monomers (Schaefer et al., Proc Natl Acad SciUSA 108:11187-92, 2011; WO 2009/080251; WO 2009/080252; WO 2009/080253).

In another embodiment, the invention provides a multimeric antibodycontaining three or more binding domains for DR4 or three or morebinding domains for DR5, e.g., four binding domains. In preferredembodiments, the anti-DR4 or anti-DR5 multimeric mAb is in theBs(scFv)₄-IgG or double variable domain format, so that it containsprecisely four binding domains for DR4 or DR5 respectively.

In especially preferred embodiments of the invention, the bispecificDR4/DR5 or multimeric anti-DR4 or anti-DR5 antibody, in any formincluding the ones specifically described above such as Bs(scFv)₄-IgGand double variable domain, comprises mutations in the constant regionthat increase binding to an Fc receptor, for example the FcγRIIbreceptor. Exemplary mutations are the single G236D, L328F, S239D, S267Emutations, and preferably double mutations G236D/S267E, S239D/S267E, andmost preferably S267E/L328F (described in S Y Chu et al., op. cit.), aswell as other mutations at these amino acid positions. Any Bs(scFv)₄-IgGor double variable domain bispecific antibody with the V1 domain bindingto DR5 and the V2 domain binding to DR4 (see FIG. 1B) with natural humanconstant regions is designated B-DR5/DR4-hFc and is designatedB-DR5/DR4-hFc** if it further contains the S267E/L328F double mutations;analogously for B-DR4/DR5-hFc and B-DR4/DR5-hFc** with V1 binding to DR4and V2 binding to DR5. A multimeric Bs(scFv)₄-IgG or double variabledomain antibody in which both V1 and V2 bind to DR4 (resp. DR5) isdesignated B-DR4/DR4-hFc (resp. B-DR5/DR5-hFc), and as B-DR4/DR4-hFc**(resp. B-DR5/DR5-hFc**) if it further contains the S267E/L328Fmutations.

The invention provides also variant antibodies including bispecificantibodies whose light and heavy chain differ from the ones specificallydescribed herein by a small number (e.g., typically no more than 1, 2,3, 5 or 10) of replacements, deletions or insertions, usually in the Cregion or V region framework but possibly in the CDRs. Most often thereplacements made in the variant sequences are conservative with respectto the replaced amino acids. Amino acids can be grouped as follows fordetermining conservative substitutions, i.e., substitutions within agroup: Group I (hydrophobic sidechains): met, ala, val, leu, ile; GroupII (neutral hydrophilic side chains): cys, ser, thr; Group III (acidicside chains): asp, glu; Group IV (basic side chains): asn, gln, his,lys, arg; Group V (residues influencing chain orientation): gly, pro;and Group VI (aromatic side chains): trp, tyr, phe. Preferably,replacements in the antibody have no substantial effect on the bindingaffinity or potency of the antibody, that is, on its ability to transmitan apoptotic signal through DR4 and/or DR5. Preferably the variantsequences are at least 90%, more preferably at least 95%, and mostpreferably at least 98% identical to the original sequences. Inaddition, other allotypes or isotypes of the constant regions may beused.

4. Therapeutic Methods

In a preferred embodiment, the present invention provides apharmaceutical formulation comprising any antibody described herein, forexample a B-DR5/DR4-hFc, B-DR5/DR4-hFc**, B-DR4/DR5-hFc orB-DR4/DR5-hFc** bispecific mAb, or B-DR4/DR4-hFc, B-DR4/DR4-hFc**,B-DR5/DR5-hFc or B-DR5/DR5-hFc** multimeric mAb, as well as humanizedforms of D114 and G4.2 such as HuD114-hFc** and HuG4.2-hFc**.Pharmaceutical formulations contain the mAb in a physiologicallyacceptable carrier, optionally with excipients or stabilizers, in theform of lyophilized or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,or acetate at a pH typically of 5.0 to 8.0, most often 6.0 to 7.0; saltssuch as sodium chloride, potassium chloride, etc. to make isotonic;antioxidants, preservatives, low molecular weight polypeptides,proteins, hydrophilic polymers such as polysorbate 80, amino acids,carbohydrates, chelating agents, sugars, and other standard ingredientsknown to those skilled in the art (Remington's Pharmaceutical Science16^(th) edition, Osol, A. Ed. 1980). The mAb is typically present at aconcentration of 0.1-1 mg/kg or 1-100 mg/ml, but most often 10-50 mg/ml,e.g., 10, 20, 30, 40 or 50 mg/ml.

In another preferred embodiment, the invention provides a method oftreating a patient with a disease by administering any antibody of theinvention, for example a B-DR5/DR4-hFc, B-DR5/DR4-hFc**, B-DR4/DR5-hFcor B-DR4/DR5-hFc** bispecific mAb, or B-DR4/DR4-hFc, B-DR4/DR4-hFc**,B-DR5/DR5-hFc or B-DR5/DR5-hFc** multimeric mAb, as well as humanizedforms of D114 and G4.2 such as HuD114-hFc** and HuG4.2-hFc**, in apharmaceutical formulation, typically in order to destroy harmful cellssuch as cancer cells expressing DR4 and/or DR5. The mAb prepared in apharmaceutical formulation can be administered to a patient by anysuitable route, especially parentally by intravenous infusion or bolusinjection, intramuscularly or subcutaneously. Intravenous infusion canbe given over as little as 15 minutes, but more often for 30 minutes, orover 1, 2 or even 3 hours. The mAb can also be injected directly intothe site of disease (e.g., a tumor), or encapsulated into carryingagents such as liposomes. The dose given is sufficient to alleviate thecondition being treated (“therapeutically effective dose”) and is likelyto be 0.1 to 5 mg/kg body weight, for example 1, 2, 3, 4 or 5 mg/kg, butmay be as high as 10 mg/kg or even 15 or 20 or 30 mg/kg, e.g., in theranges 0.1-1 mg/kg, 1-10 mg/kg or 1-20 mg/kg. A fixed unit dose may alsobe given, for example, 50, 100, 200, 500 or 1000 mg, or the dose may bebased on the patient's surface area, e.g., 1000 mg/m². Usually between 1and 8 doses, (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) are administered to treatcancer, but 10, 20 or more doses may be given. The mAb can beadministered daily, twice per week, weekly, every other week, monthly orat some other interval, depending, e.g. on the half-life of the mAb, for1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3-6 months or longer.Repeated courses of treatment are also possible, as is chronicadministration.

Diseases especially susceptible to therapy with the mAbs of thisinvention include those associated with cells such as cancer cellsexpressing elevated levels of DR4 and/or DR5, compared with normal cellsof the same tissue type, for example ovarian cancer, breast cancer, lungcancer (small cell or non-small cell), colon cancer, prostate cancer,pancreatic cancer, gastric cancer, liver cancer (hepatocellularcarcinoma), kidney cancer (renal cell carcinoma), head-and-neck tumors,melanoma, sarcomas, and brain tumors (e.g., glioblastomas). Hematologicmalignancies such as leukemias and lymphomas may also be susceptible.Optionally expression of DR4 and/or DR5 in cancer cells of a subjectbeing treated can be assessed before initiating or continuing treatment.Expression can be assessed at the mRNA or preferably at the proteinlevel, for example by immune assay of biopsy specimens, such asimmunohistochemistry (IHC). Expression of DR4 and/or DR5 is preferablyat least above background assessed with an irrelevant control antibodyby immune assay and more preferably above the level of noncancerouscells of the same tissue type. Other diseases susceptible to therapywith the mAbs of the invention include autoimmune diseases such asrheumatoid arthritis, multiple sclerosis and inflammatory bowel disease.

In a preferred embodiment, the mAb of the invention is administered incombination with (i.e., together with, that is, before, during or after)other therapy. For example, to treat cancer, the mAb may be administeredtogether with any one or more of the known chemotherapeutic drugs, forexample alkylating agents such as carmustine, chlorambucil, cisplatin,carboplatin, oxaliplatin, procarbazine, and cyclophosphamide;antimetabolites such as fluorouracil, floxuridine, fludarabine,gemcitabine, methotrexate and hydroxyurea; natural products includingplant alkaloids and antibiotics such as bleomycin, doxorubicin,daunorubicin, idarubicin, etoposide, mitomycin, mitoxantrone,vinblastine, vincristine, and Taxol (paclitaxel) or related compoundssuch as Taxotere®; the topoisomerase 1 inhibitor irinotecan; andinhibitors of tyrosine kinases such as Gleevec® (imatinib), Sutent®(sunitinib), Nexavar® (sorafenib), Tarceva® (erlotinib), Tykerb®(lapatinib), Iressa® (gefitinib) and Xalkori® (crizotinib); Rapamycin®(sirolimus) and other mTOR inhibitors; and inhibitors of angiogenesis;and all approved and experimental anti-cancer agents listed in WO2005/017107 A2 (which is herein incorporated by reference). The mAb maybe used in combination with 1, 2, 3 or more of these other agents,preferably in a standard chemotherapeutic regimen. Normally, the otheragents are those already believed or known to be effective for theparticular type of cancer being treated.

Other agents with which the mAb of the invention can be administered totreat cancer include biologics such as monoclonal antibodies, includingHerceptin® or Perjeta® (pertuzumab), against the HER2 antigen; Avastin®against VEGF; or antibodies to the Epidermal Growth Factor (EGF)receptor such as Erbitux® (cetuximab) and Vectibix® (panitumumab),activators of the immune system such as Yervoy® (ipilimumab) includinganti-PD-1 mAbs such as Opdivo® (nivolumab) and Keytruda®(pembroluzimab), as well as antibody-drug conjugates such as Kadcyla™(ado-trastuzumab emtansine). Moreover, the mAb can be used together withany form of surgery and/or radiation therapy.

Treatment (e.g., standard chemotherapy) including the mAb of theinvention may increase the median progression-free survival or overallsurvival time of patients with a particular type of cancer such as thoselisted above by at least 20% or 30% or 40% but preferably 50%, 60% to70% or even 100% or longer, compared to the same treatment (e.g.,chemotherapy) but without the mAb; or by (at least) 2, 3, 4, 6 or 12months. In addition or alternatively, treatment (e.g., standardchemotherapy) including the mAb may increase the complete response rate,partial response rate, or objective response rate (complete+partial) ofpatients (especially when relapsed or refractory) by at least 30% or 40%but preferably 50%, 60% to 70% or even 100% compared to the sametreatment (e.g., chemotherapy) but without the mAb.

Typically, in a clinical trial (e.g., a phase II, phase II/III or phaseIII trial), the aforementioned increases in median progression-freesurvival and/or response rate of the patients treated with chemotherapyplus the mAb of the invention, relative to the control group of patientsreceiving chemotherapy alone (or plus placebo), is statisticallysignificant, for example at the p=0.05 or 0.01 or even 0.001 level. Itis also understood that response rates are determined by objectivecriteria commonly used in clinical trials for cancer, e.g., as acceptedby the National Cancer Institute and/or Food and Drug Administration,for example the RECIST criteria (Response Evaluation Criteria In SolidTumors).

5. Other Methods

The mAbs of the invention also find use in diagnostic, prognostic andlaboratory methods. They may be used to measure the level of DR4 and/orDR5 in a tumor or in the circulation of a patient with a tumor, andtherefore to follow and guide treatment of the tumor. For example, atumor associated with high levels of DR4 and/or DR5 would be especiallysusceptible to treatment with the mAb. In particular embodiments, themAbs can be used in an ELISA or radioimmunoassay to measure the level ofDR4 and/or DR5, e.g., in a tumor biopsy specimen or in serum. The use ofone anti-DR4 mAb and one anti-DR5 mAb is especially useful to detectcells that express both DR4 and DR5. For various assays, the mAb may belabeled with fluorescent molecules, spin-labeled molecules, enzymes orradioisotopes, and may be provided in the form of kit with all thenecessary reagents to perform the assay. In other uses, the mAbs areused to purify DR4 or DR5, e.g., by affinity chromatography.

6. Examples Example 1 Generation of Anti-DR4 and Anti-DR5 mAbs

To generate and assay mAbs that bind to human DR4, several fusionproteins were constructed using standard methods of molecular biology.To produce DR4-hFc, cDNA encoding the extracellular domain (amino acids109 to 239) of human DR4 was generated and inserted into derivatives ofthe pCI vector (Invitrogen), linked to a human Ig gamma-1 Fc region(hinge-cH2-cH3) at the C-terminus, transfected and expressed in 293Fmammalian cells. A cynomolgus monkey cDR4-hFc fusion protein wassimilarly constructed using the monkey DR4 extracellular domain clonedfrom cynomolgus monkey liver cDNA (Biochain) to obtain the monkey DR4domain. These Fc-fusion proteins were purified from 293F culturesupernatant by using a protein A column. The DR4-KF fusion protein wassimilarly produced by linking the human DR4 extracellular domain to thehuman Ig kappa constant region followed by the FLAG peptide (DYKDDDDK)(SEQ ID NO:41) at the C-terminus; an analogous protein cDR4-KF was madeusing cynomolgus monkey DR4. These FLAG-tagged fusion proteins werepurified using anti-FLAG columns. To generate and assay mAbs that bindto human DR5, a completely analogous set of proteins DR5-hFc and DR5-KF,and cDR5-hFc and cDR5-KF, were made in the same manner as for DR4 usingthe human and cynomolgus monkey DR5 extracellular domain (amino acids54-183) respectively. For blocking assays, human Apo2L/TRAIL protein(amino acids 95-281) was used (R&D systems). Commercial human DR4-Fc andDR5-Fc fusion proteins (R&D systems) were also used.

To generate anti-DR4 mAbs, Balb/c mice were immunized in each hindfootpad twice weekly 11 times with 1 μg DR4-hFc (TRAILR1-Fc from R&DSystems) and then 5 times with 5 μg DR4-hFc prepared as described above,resuspended in MPL/TDM adjuvant (Sigma-Aldrich). Three days after thefinal boost, popliteal lymph node cells were fused with murine myelomacells, P3X63AgU.1 (ATCC CRL1597), using 35% polyethylene glycol.Hybridomas were selected in HAT medium as described (Chuntharapai andKim, J Immunol 163:766, 1997). Ten days after the fusion, hybridomaculture supernatants were screened in the DR4 binding ELISA describedbelow. Selected hybridomas were cloned twice by limiting dilution. MAbsin ascites and culture fluids of selected hybridomas were purified usinga protein A/G column. Of several antibodies selected for furtheranalysis, the antibody D114 was chosen for development because of itssuperior agonist activity shown in cell killing assays. The isotype ofD114 was determined to be IgG1, kappa using an isotyping kit.

To generate anti-DR5 mAbs, Balb/c mice were immunized in each hindfootpad twice weekly 12 times with 5 μg DR5-hFc and then once with 1 μgDR5-hFc resuspended in MPL/TDM adjuvant (Sigma-Aldrich). Three daysafter the final boost, popliteal lymph node cells were fused with murinemyeloma cells and hybridomas selected in HAT medium as described abovefor anti-DR4 mAbs. Ten days after the fusion, hybridoma culturesupernatants were screened in the DR5 binding ELISA described below.Selected hybridomas were then cloned twice by limiting dilution and themAbs purified as described above for anti-DR4 mAbs. Selected mAbs werefurther tested for their effects on COLO 205 cell viability and fortheir binding activity to monkey DR5 as described below. Of severalselected antibodies, the antibody G4.2 was chosen for developmentbecause of its superior agonist activity shown in cell killing assays.The isotype of G4.2 was determined to be IgG1, kappa using an isotypingkit.

Example 2 Binding and Blocking Activity of Antibodies

Each step of each ELISA assay described in this patent application wasperformed by room temperature incubation with the appropriate reagentfor 1 hour, except the initial plate coating step was done overnight at4° C. followed by blocking with 2% BSA for 1 hr. Between each step,plates were washed 3 times in PBS containing 0.05% Tween 20. Data pointswere generally in duplicate or triplicate; there was generally littlevariability between replicate data points.

To measure binding of mAbs to DR4 (respectively DR5), capture assayswere generally used: plates were first coated with goat anti-mouseIgG-Fc (2 μg/mL) overnight, blocked with 2% BSA, incubated withhybridoma supernatant or increasing concentrations of purified antibodyto be tested, and then with 0.3 μg/ml of DR4-KF (resp. DR5-KF). TheDR4-KF (resp. DR5-KF) captured was detected by addition of HRP-goatanti-human kappa, and then TMB substrate. However, to measure binding ofthe HuG4.2 variants to DR5 as described below, a direct binding assaywas used: plates were coated with Dr5-Fc (0.5 μg/mL), blocked, incubatedwith increasing concentrations of mAbs, and bound mAb detected withHRP-goat anti-human kappa.

To measure blocking activity of mAbs, plates were first coated with goatanti-human IgG-Fc (2 μg/ml) overnight, blocked with 2% BSA and thenincubated with DR4-hFc or resp. DR5-hFc (0.5 μg/ml). Then 50 ng/ml Apo2L(TRAIL, R&D Systems) mixed with various concentrations of purified mAbwere added to the wells, and the bound Apo2L was detected by theaddition of 0.2 μg/ml of biotinylated anti-TRAIL antibody (R&D Systems),followed by the addition of HRP-Streptavidin and then TMB substrate.

Using these ELISA assays, it was shown that D114 binds to DR4 as well as4H6 does (FIG. 2A), and blocks the binding of Apo2L to DR4 as well as4H6 does (FIG. 2B). It was similarly shown that D114 binds to cynomolgusmonkey DR4 about as well as to human DR4 by using the cDR4-hFc proteinin the binding assay. It was also shown that G4.2 binds well to DR5(FIG. 2C) and effectively blocks the binding of Apo2L to DR5 (FIG. 2D).Moreover, it was shown that G4.2 also binds well to cynomolgus monkeyDR5 by using the cDR5-hFc protein in the binding assay described above,whereas 3H3 does not bind to cynomolgus DR5 in this assay, providing anadvantage of G4.2 relative to 3H3 and showing that G4.2 must have adifferent epitope than 3H3.

Example 3 Cell Killing by D114 and G4.2

To perform assays to measure reduction of cell viability (called cellkilling herein), human tumor cell lines in DMEM/10% FCS were plated intoa 96-well plate at 2×10⁴ cells/100 μl/well and incubated overnight (instandard conditions of 37° C. and 5% CO₂). The media was then removedand replaced by 100 μl of the same media containing increasingconcentrations of mAb, with or without 10 μg/mL goat anti-mouse IgG-Fc(anti-mIgG-Fc) for mouse antibodies and goat anti-human IgG-Fc(anti-hIgG-Fc) for human, humanized or chimeric antibodies. In certainassays, the mAb and anti-IgG-Fc were instead included in the media whenthe tumor cells were plated. Each concentration of mAb was done induplicate. After incubation overnight in standard conditions, the cellviability was determined by the addition of 10 μl/well of WST-8 for 1 hrand measurement of absorbance at OD 450. The purpose of the goatanti-mIgG-Fc or anti-hIgG-Fc used in some experiments was to cross-link(oligomerize) the anti-DR4 or anti-DR5 antibody being tested, thuspotentially transmitting a stronger apoptotic signal to the cells. Suchcross-linking by anti-IgG-Fc antibody is believed to mimic thecross-linking that occurs when the anti-DR4 or anti-DR5 antibody bindsto and is thus linked by white blood cells infiltrating a tumor via FcγRon the surface of such cells.

Using this assay, it was determined that D114 inhibits cell viability ofthe H460 lung tumor cell line (ATCC HTB-177; FIG. 3A) and the SW480colon tumor cell line (ATCC CCL-228; FIG. 3B) and other tested celllines as well as 4H6, in the presence of anti-mIgG-Fc. Similarly, G4.2inhibits cell viability of H460 cells (FIG. 3C) and COLO 205 colon tumorcells (ATCC CCL-222; FIG. 3D) almost as well as 3H3 does.

Example 4 Construction and Characterization of Antibodies

Cloning of the heavy and light chain variable regions of the mAbs,construction and expression of various mAbs, and introduction ofmutations were all performed using standard methods of molecularbiology, e.g. as described in U.S. Pat. No. 7,632,926, which is hereinincorporated by reference for all purposes. The amino acid sequences ofthe (mature) heavy and light chain variable regions of 4H6 are shownrespectively in FIGS. 4A and 4B and the (mature) heavy and light chainvariable regions of 3H3 are shown respectively in FIGS. 4C and 4D. The(mature) heavy and light chain variable regions of D114 are shownrespectively in FIGS. 5A and 5B, top lines labeled D114; and the(mature) heavy and light chain variable regions of G4.2 are shownrespectively in FIGS. 5C and 5D, top lines labeled G4.2.

In addition, for comparison to the mAbs disclosed herein, several otheranti-DR5 mAbs were constructed based on their published sequences: thehuman mAbs drozitumab (Apomab; FIGS. 17 and 18 of U.S. Pat. No.8,030,023) and conatumumab (AMG 655; FIG. 19 of WO 2012/106556), and themouse mAb TRA-8 (FIGS. 23 and 24 of U.S. Pat. No. 7,244,429).

As a convention used herein, the hFc** suffix (also written as Fc** incertain figures) applied to an antibody means it contains the humangamma-1 constant region with S267E and L328F mutations, the sequence ofwhich is shown as part of FIG. 6A (see legend to FIG. 6A), whereas thehFc suffix means it contains the human gamma-1 constant region withoutthe S267E and L328F mutations (thus having S and L at those amino acidpositions). We constructed 3H3-hFc, 4H6-hFc, D114-hFc and G4.2-hFcchimeric antibodies by combining the variable regions of the respectivemouse antibodies with a human kappa constant region and human gamma-1constant region, and the corresponding 3H3-hFc**, 4H6-hFc**, D114-hFc**and G4.2-hFc** chimeric antibodies with the human gamma-1 constantregion containing the S267E and L328F mutations. For comparison in someexperiments, we also constructed the antibody Apomab-hFc** by combiningthe human Apomab variable regions with human gamma-1 constant regioncontaining the S267E and L328F mutations

A bispecific antibody B-4H6/3H3-hFc** in the Bs(scFv)₄-IgG format wasconstructed: the amino acid sequences of the heavy and light chains ofthis mAb are shown respectively in FIG. 6A and FIG. 6B, with each maturechain starting at the first amino after the signal peptide. Hence, thismAb has the configuration shown in FIG. 1A with V_(H)1 and V_(L)1 fromthe 4H6 mAb and V_(H)2 and V_(L)2 from the 3H3 mAb.

Example 5 Cell Killing by Antibodies

The ability of various antibodies described above to kill different celllines (i.e., reduce cell viability) was tested in the cell killing assaydescribed above. In the absence of anti-hIgG-Fc, neither 3H3-hFc** nor4H6-hFc** nor even a combination (mixture) of these two mAbs showed anykilling of either the SW480 colon tumor cells (FIG. 7A) or H460 lungtumor cells (FIG. 8A), because without a cross-linking agent tooligomerize the antibodies, they cannot transmit a death signal to thecells. However, 3H3-hFc* and to an even greater extent 4H6-hFc** couldkill the SW480 cells in the presence of the cross linking agentanti-hIgG-Fc (FIG. 7B); adding 3H3-hFc** to 4H6-hFc** did not furtherincrease killing.

To determine whether the antibody forms with mutations would have anadvantage in killing tumor cells in the presence of FcγRIIb-expressingcells, peripheral blood mononuclear cells (PBMCs) consisting largely ofmonocytes and lymphocytes were isolated from human blood usingFicoll-Paque PLUS (GE Healthcare) according to the manufacturer'sinstructions. The cell viability assays were performed as describedabove, except the media containing mAbs also contained generally 2×10⁵human PBMCs. In the presence of the PBMCs, 4H6-hFc** in fact killed H460tumor cells substantially more effectively than 4H6-hFc (FIG. 8B)

We next explored whether two mutations in the human gamma-1 constantregion provided greater cell killing activity than a single mutation. Wethus constructed the chimeric 4H6-hFc* mAb (denoted by hFc* with asingle asterisk) having only the S267E mutation, rather than theS267E/L328F double mutation in 4H6-hFc**. The cell killing assaydescribed above was used with a fixed concentration of 0.5 μg/mL mAb,4×10⁴ COLO 205 tumor cells per well and 4×10⁵ PBMCs per well from fourdifferent human donors, to take into account potential variability ofPBMCs. For each of the donors, 4H6-hFc modestly reduced viability of thetumor cells (relative to control hIgG mAb set to 100% viability for eachdonor), 4H6-hFc* with single mutation reduced viability slightly tosignificantly more than 4H6-hFc, but 4H6-hFc** reduced viabilitysubstantially better than 4H6-hFc* (FIG. 9A), showing the advantage oftwo mutations that increase binding to FcγRIIb over one such mutation.To determine whether this was also true of other combinations of atleast two mutations, we introduced other pairs of mutations into 4H6-hFcaccording to the following table:

TABLE 1 Mutation Variants Designation 1st mutation 2nd mutation 3^(rd)mutation 4H6-hFc** (2) S267E L328F — 4H6-hFc** (3) S267E S239D —4H6-hFc** (4) I332E S239D — 4H6-hFc*** (5) I332E S239D A330L

At a fixed antibody concentration of 1 μg/mL and in the presence ofhuman PBMCs, all four variants reduced cell viability approximatelyequally for COLO 205 cells, MDA-MB-231 cells (ATCC HTB-26) and H460cells (FIG. 9B), with the greatest effect seen on the COLO 205 cells andonly a modest effect seen for H60 under the conditions of the assay.Hence, various combinations of two or more mutations that each increasebinding to FcγRIIb are equally suitable to enhance tumor cell killing.

To further explore the advantage of two mutations over one mutation, theability of 4H6-hFc, 4H6-hFc*, and 4H6-hFc** to kill COLO 205 cells inthe presence of PBMCs was tested over a range of antibody concentrations(FIG. 10A). No killing by 4H6-hFc was seen in this experiment, modestkilling by 4H6-hFc*, and substantial killing by 4H6-hFc**, again showingthe advantage of two over one mutation.

As a final experiment in this regard, the ability of 4H6-hFc, 4H6-hFc*,and 4H6-hFc**, inhibit growth of COLO 205 tumor xenografts in mice wascompared, using the methods described just below. When 2 mg/kg of eachmAb was administered twice per week, 4H6-hFc** with two mutationsinhibited the xenografts more strongly than either 4H6-Fc or 4H6-Fc*(FIG. 10B) with respectively no and one mutation, consistent with thecell killing results.

Example 6 Ability of Antibodies to Inhibit Growth of Tumor Xenografts

Xenograft experiments were carried out essentially as describedpreviously (Kim et al., Nature 362:841, 1993). Human tumor cellstypically grown in complete DMEM medium were harvested in HBSS. Femaleimmunodeficient mice (4-6 weeks old) were injected subcutaneously with2-10×10⁶ cells in 0.1-0.2 ml of HBSS, with Matrigel (Corning) in someexperiments, in the dorsal areas. When the tumor size reached about 100mm³, the mice were grouped randomly, and typically 0.5-5 mg/kg of mAbswere administered i.p. once only or once or twice per week in a volumeof 0.1 ml. Tumor sizes were determined twice a week by measuring in twodimensions [length (a) and width (b)]. Tumor volume was calculatedaccording to V=ab²/2 and expressed as mean tumor volume±SEM. The numberof mice in each treatment group was typically 5-7 mice. Statisticalanalysis can be performed, e.g., using Student's t test on the finaldata point.

We first conducted xenograft experiments to determine whether,consistent with the enhanced cell killing in the presence of PBMCsdescribed above, the hFc** forms of anti-DR4 and anti-DR5 mAbs were moreeffective than the hFc forms in vivo, due to enhanced FcγR bindingprovided by the mutations in the hFc** constant region. Indeed, whereasthe anti-DR4 mAb D114-hFc only moderately inhibited growth of COLO 205xenografts when administered as a single dose of 2 mg/kg, D114-hFc**almost completely inhibited the xenografts under these conditions (FIG.11A). (As described below, very similar results were obtained forinhibition of COLO 205 xenografts by the humanized forms, HuD114-hFc andHuD114-hFc**; FIG. 18A). And whereas D114-hFc did not significantlyinhibit growth of SW480 colon tumor xenografts when also administered inthis manner, D114-hFc** moderately inhibited xenograft growth underthese conditions (FIG. 11B). Finally, another anti-DR4 mAb 4H6-hFcmoderately inhibited growth of COLO 205 xenografts when given at 2 mg/kgtwice per week, but 4H6-hFc** completely inhibited xenografts underthese conditions (FIG. 11C).

Regarding anti-DR5 mAbs, 3H3-Fc did not significantly inhibit growth ofCOLO 205 xenografts when administered once at 0.5 mg/kg, but 3H3-hFc**completely inhibited growth of these xenografts even when given at thisvery low dose level (FIG. 12A). Similarly, 3H3-Fc only modestlyinhibited growth of MIA PaCa-2 (ATCC CRL-1420) pancreatic tumorxenografts when administered once at 1 mg/kg, but 3H3-hFc** completelyinhibited growth of these xenografts even when given at this very lowdose level (FIG. 12B). Analogously, as described below, whenadministered once at 1 mg/kg, the humanized mAb HuG4.2-hFc** inhibitedgrowth of both COLO 205 xenografts and MIA PaCa-2 xenografts better thanHuG4.2-hFc did (FIGS. 19A, B). Thus for both anti-DR4 and anti-DR5 mAbs,the hFc** forms with mutations were much more effective in vivo than thehFc forms without mutations.

Example 7 Construction and Characterization of Humanized Antibodies

Design, construction, expression and purification of humanized D114 andhumanized G4.2 mAbs were all performed using standard methods ofmolecular biology, e.g. as described in U.S. Pat. No. 7,632,926 for theL2G7 mAb, which is herein incorporated by reference for all purposes.More specifically, to design a humanized D114 (respectively G4.2) mAb,the methods of Queen et al., U.S. Pat. Nos. 5,530,101 and 5,585,089 weregenerally followed. The human VK sequence AIT38746 and VH sequenceAAC18293 (respectively human VK sequence AAQ02698 and VH sequenceAAC50998), as shown in FIGS. 5A and 5B (resp. 5C and 5D), bottom lines,were respectively chosen to serve as acceptor sequences for the D114(resp. G4.2) VL and VH sequences, because they have particularly highframework homology (i.e., sequence identity) to them. Acomputer-generated molecular model of the D114 (respectively G4.2)variable domain was used to locate the amino acids in the framework thatare close enough to the CDRs to potentially interact with them. Todesign the humanized D114 (respectively G4.2) light and heavy chainvariable regions, the CDRs from the mouse D114 (resp. G4.2) mAb werefirst conceptually grafted into the acceptor framework regions. Atframework positions where the computer model suggested significantcontact with the CDRs, which may be needed to maintain the CDRconformation, the amino acids from the mouse antibody were substitutedfor the human framework amino acids.

For D114, such substitutions were made at residues 48 and 71 of thelight chain (HuD114-L1), and at residues 48 and 71 of the heavy chain(HuD114-H1) or at these residues plus the additional heavy chainresidues 67 and 69 (HuD114-H2). The light and heavy chain V regionsequences of humanized D114 are shown in FIGS. 5A and 5B respectively,middle lines labeled HuD114, where they are aligned against therespective D114 donor and human acceptor V regions. The mAbs with theHuD114-L1 light chain and HuD114-H1 or HuD114-H2 heavy chain arerespectively designated HuD114 #1 and HuD114 #2. Each of these was madein two forms: HuD114-hFc where the human gamma-1 constant region doesnot have the S267E and L328F mutations (and thus has S and L atpositions 267 and 328), and HuD114-hFc** where the constant region doeshave these mutations. Using the binding assay described above, bothHuD114-hFc #1 and HuD114-hFc #2 bind to DR4 as well as the chimericantibody D114-hFc described above (FIG. 13A), indicating that noaffinity was lost during humanization. HuD114-hFc** #1 and HuD114-hFc**#2 kill COLO 205 cells in the presence of goat anti-hIgG-Fc equally well(FIG. 14A). In an attempt to improve cell killing activity, new versionsof the HuD114 light and heavy chain were produced having additionalmouse substitutions: HuD114-L2, which contains mouse substitutions atresidues 48 and 71 plus 43 and 44, and HuD114-H3, which contains mousesubstitutions at residues 48, 67, 69 and 71 plus 91. Six antibodiesconsisting of all combinations of HuD114-L1 and HuD114-L2 withHuD114-H1, HuD114-H2 and HuD114-H3 were produced, but they all hadcomparable binding activity and cell killing activity. Hence, HuD114-hFc#2 and HuD114-hFc** #2 were used for further studies and are denotedHuD114-hFc and HuD114-hFc** henceforward and in the figures.

The invention also includes variants of these preferred antibodies, forexample humanized antibodies comprising a light chain V region with asequence at least 90, 95, 98 or 99% identical to HuD114-L1 in FIG. 5Aand a heavy chain V region with a sequence at least 90, 95, 98 or 99%identical to HuD114-H1 or HuD114-H2 in FIG. 5B. Preferably all CDRresidues in such antibodies are those of the donor. Preferably, lightchain positions 48 and 71 are occupied by V and Y respectively and heavychain positions 48 and 71 are occupied by L and A respectively.

Similarly, for G4.2 mouse substitutions were made at residues 4 and 68of the light chain (HuG4.2-L1) or at these residues plus residue 1(HuG4.2-L2), and at residues 27, 28, 30 and 93 of the heavy chain(HuG4.2-H1) or at these residues plus residue 47 (HuG4.2-H2). The lightand heavy chain V region sequences of HuG4.2 are shown in FIGS. 5C and5D respectively, middle lines labeled HuG4.2, where they are alignedagainst the respective G4.2 donor and human acceptor V regions. Bycombining each of the humanized light chains with each of the humanizedheavy chains, four different humanized G4.21 antibodies designatedHuG4.2 #1, #2, #3 and #4 were made, as shown in the following table,where the number of substitutions in each chain is given in parentheses.As for HuD114, each of these mAbs was made in two forms: hFc withoutmutations in the human gamma-1 heavy chain constant region, and hFc**with the mutations S267E and L328F.

TABLE 2 HuG4.2 Variants HuG4.2 Light Chain Heavy Chain #1 L1 (2) H1 (4)#2 L1 (2) H2 (5) #3 L2 (3) H1 (4) #4 L2 (3) H2 (5)

All four versions of HuG4.2 bound well to DR5, with HuG4.2-hFc** #1 andHuG4.2-hFc** #2 slightly better than HuG4.2-hFc** #3 and HuG4.2-hFc** #4(FIG. 13B). Indeed, HuG4.2-hFc** #1 and #2 bound to DR5 comparably tothe chimeric antibody G4.2-hFc**, indicating that little or no affinitywas lost during humanization. All four versions of HuG4.2 also killedCOLO 205 cells well in the presence of goat anti-hIgG-Fc (FIG. 14B),with HuG4.2-hFc** #2 slightly better than the others and essentially thesame as the chimeric antibody G4.2-hFc**. Hence, HuG4.2-Fc #2 andHuG4.2-hFc** #2 were used for further studies, and are denoted HuG4.2-Fcand HuG4.2-Fc** henceforward and in the figures.

The invention also includes variants of HuG4.2 which comprise a lightchain V region with a sequence at least 90, 95, 98 or 99% identical toHuG4.2-L1 or HuG4.2-L2 in FIG. 5C and a heavy chain V region with asequence at least 90, 95, 98 or 99% identical to HuG4.2-H1 or HuG4.2-H2in FIG. 5D. Preferably all CDR residues in such antibodies are those ofthe donor antibody. Preferably light chain positions 4 and 68 areoccupied by L and R respectively, and heavy chain positions 27, 28, 30and 93 are occupied by L, P, N and T respectively. Optionally, heavychain position 47 is occupied by L.

The ability of the various humanized mAbs to kill cells was tested inthe presence of goat anti-IgG-Fc (10 μg/mL) as cross-linking agent. BothHuD114-hFc and HuD114-hFc** killed COLO 205 colon tumor cells (FIG. 15A)and SW480 colon tumor cells (FIG. 15B) much better than did mapatumumab,the anti-DR4 mAb that has been tested in clinical trials. However, therewas no significant difference between HuD114-hFc and HuD114-hFc**, asexpected because the increase in binding to FcγRIIb provided by themutations should not have an effect in the absence of FcγRIIb-expressingcells. Similarly, the ability of HuG4.2-hFc and/or HuG4.2-hFc** wascompared with that of the anti-DR5 mAbs Apomab, AMG 655 and lexatumumabto kill COLO 205 cells (FIG. 15C), and with Apomab and TRA-8 to kill MIAPaCa-2 pancreatic tumor cells (FIG. 15D). Again, where tested, there wasno significant difference between HuG4.2-hFc and HuG4.2-hFc** because ofthe absence of FcγRIIb-expressing cells. However, HuG4.2-hFc andHuG4.2-hFc** killed both cell lines substantially better than the othertested mAbs, which have been in clinical trials. The ability of variousmAbs to kill H460 cells (FIG. 16A) and SW480 cells (FIG. 16B) in thepresence of anti-hIgG-Fc was also determined. HuD114-hFc** andHuG4.2-hFc** killed H460 cells substantially better than the anti-DR5mAbs Apomab and AMG 655, while HuD114-hFc** killed SW480 cellssubstantially better and HuG4.2-hFc** slightly better than Apomab andAMG655, consistent with the results just noted (FIGS. 15A-D).

Next, we compared the ability of various mAbs to kill cells in thepresence of human PBMCs, where the hFc** forms might be superior to thehFc forms due to the enhanced FcγRIIb binding provided by the S267E andL328F mutations. Indeed, HuG4.2-Fc** killed either COLO 205 cells (FIG.17A) or H60 cells (FIG. 17B) much better than HuG4.2-Fc, and likewiseApomab-hFc**, a form of Apomab in which we introduced these mutations asdescribed above, killed the cells better than Apomab itself (FIGS. 17A,B). However, HuG4.2-hFc** still killed either COLO 205 or H60 cellssubstantially better than Apomab-hFc** (and better than AMG 655), againshowing the superior potency of the HuG4.2-hRFc** anti-DR5 mAb.

To compare the effect of two versus one mutation in the context ofHuD114 and HuG4.2, we tested the cell-killing ability in the presence ofPBMCs of the mAbs HuD114-hFc (respectively HuG4.2-hFc) withoutmutations, the mAbs HuD114-hFc* (resp. HuG4.2-hFc*) with only the S267Emutation, and the mAbs HuD114-hFc** (resp. HuG4.2-hFc**) with theS267E/L328F double mutation. HuD114-hFc* killed COLO 205 cells (FIG.17C) and H460 cells (FIG. 17D) slightly better than HuD114-hFc did,while HuD114-hFc** killed the cells much better. Similarly, HuG4.2-hFc*killed COLO 205 cells (FIG. 17E) and H460 cells (FIG. 17F) slightlybetter than HuG4.2-hFc did, while HuG4.2-hFc** killed the cells muchbetter. Hence the presence of two mutations in the constant region issubstantially advantageous over one mutation.

Turning finally to the ability of the humanized mAbs HuD114 and HuG4.2to inhibit growth of xenografts, HuD114-hFc moderately inhibited thegrowth of COLO 205 xenografts when administered once at 2 mg/kg, whileHuD114-hFc** completely inhibited xenograft growth under theseconditions (FIG. 18A). HuD114-hFc** given once at 2 mg/kg also stronglyinhibited growth the ability of H460 lung tumor xenografts (FIG. 18B).In another experiment, at the low dose tested, HuD114-hFc* completelyinhibited growth of COLO 205 xenografts, while the antibody mapatumumab,which has been tested in clinical trials, had no effect (FIG. 18C). Whenadministered once at 2 mg/kg, HuD114-hFc** was also somewhat moreeffective than mapatumumab in inhibiting growth of Ramos (ATCC CRL-1596)lymphoma xenografts (FIG. 18D). A single dose of 1 mg/kg of HuG4.2-hFcor HuG4.2-hFc** inhibited growth of COLO 205 xenografts (FIG. 19A) andMIA PaCa-2 pancreatic tumor xenografts (FIG. 19B), with HuG4.2-hFc**clearly more effective than HuG4.2-hFc. (HuD114-hFc** was also effectivein this model; FIG. 19B). Greater efficacy of the hFc** forms of thesemAbs relative to the hFc forms in xenograft models is consistent withthe greater ability of the hFc** forms to kill tumor cells in vitro inthe presence of PBMCs described above.

Example 8 Characterization of a Bispecific Antibody

To show that the bispecific B-4H6/3H3-hFc** mAb described above (FIGS.6A, B) can simultaneously bind to DR4 and DR5, plates were first coatedovernight with an anti-DR4 mAb (2 μg/ml) that does not compete forbinding with 4H6, blocked with 2% BSA and then incubated with DR4-hFc(0.5 μg/ml). Then increasing concentrations of B-4H6/3H3-hFc** mAb wereadded to the wells, followed by 0.5 μg/ml of DR5-KF. Bound DR5-KF wasdetected with HRP-anti-Flag M2 (Sigma) and substrate. As only a mAb thatcan bind to both DR4 (in the form of DR4-hFc) and DR5 (in the form ofDR5-KF) will give a positive result in this ELISA assay, the results(FIG. 20A) show that B-4H6/3H3-hFc** has this capability, whereas ofcourse the mAbs 4H6-hFc** and 3H3-hFc** do not.

The ability of B-4H6/3H3-hFc** to kill COLO 205 without thecross-linking agent anti-mIgG-Fc was determined. 4H6-hFc** was not ableto kill the cells in these conditions, while 3H3-hFc** exhibited somekilling (FIG. 20B). This is due to the fact that COLO 205 cells are verysensitive to death receptor agonists, more so than other cell lines suchas H60 and SW480 utilized herein. However, the B-4H6/3H3-hFc**bispecific mAb killed the cells substantially better than 3H3-hFc**,although the 4H6 component had no cell killing ability on its own (FIG.20B). In contrast, simply mixing the separate mAbs 3H3-hFc** and4H6-hFc** did not increase the cell killing ability of either one (FIGS.7A, B).

Another bispecific antibody B-HuD114/HuG4.2-hFc** was constructed in theBs(scFv)4-IgG format in the same manner as B-4H6/3H3-hFc**, and alsocontaining the double mutation in the constant region. Using the ELISAassay described above in this example, this B-HuD114/HuG4.2-hFc** mAbbound to both DR4 and DR5, while HuG4.2-hFC** did not (FIG. 20C). AB-HuD114/HuG4.2-hFc** mAb also killed COLO 205 cells in the absence ofanti-hIgG-Fc (FIG. 20D), even better than the natural ligand TRAIL did.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the invention. Unlessotherwise apparent from the context any step, element, embodiment,feature or aspect of the invention can be used with any other. Allpublications, patents and patent applications including accessionnumbers and the like cited are herein incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent and patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes. The word “herein” indicates anywhere in this patentapplication, not merely within the section where the word “herein”occurs. If more than one sequence is associated with an accession numberat different times, the sequence associated with the accession number asof the effective filing date of this application is intended, theeffective filing date meaning the actual filing date or earlier date ofa filing of a priority application disclosing the accession number inquestion.

We claim:
 1. A monoclonal antibody (mAb) that binds to human death receptor 4 and which comprises a light chain variable (V) region having three CDRs from the light chain V region sequence of D114 (SEQ ID NO:5) and a heavy chain V region having three CDRs from the heavy chain V region sequence of D114 (SEQ ID NO:8).
 2. The mAb of claim 1 which is a humanized antibody.
 3. The mAb of claim 2 which comprises a light chain V region with a sequence at least 90% identical to HuD114-L1 (SEQ ID NO:6) and a heavy chain V region with a sequence at least 90% identical to HuD114-H1 (SEQ ID NO:9) or HuD114-H2 (SEQ ID NO:10), wherein light chain positions 48 and 71 by Kabat numbering are occupied by V and Y respectively and heavy chain positions 48 and 71 by Kabat numbering are occupied by L and A respectively.
 4. The mAb of claim 2 which comprises a light chain V region with the sequence of HuD114-L1 (SEQ ID NO:6) and a heavy chain V region with the sequence of HuD114-H1 (SEQ ID NO:9) or HuD114-H2 (SEQ ID NO:10).
 5. The mAb of claim 1 which inhibits growth of a human tumor xenograft in a mouse.
 6. The mAb of claim 1 which is a bispecific antibody.
 7. The mAb of claim 1 comprising a human constant region having one or more mutations that increase binding to a human Fc gamma receptor.
 8. The mAb of claim 7 wherein the human constant region is the gamma-1 constant region and the one or more mutations comprise one or both of the S267E and L328F mutations.
 9. A pharmaceutical composition comprising the antibody of claim 1 in a pharmaceutically acceptable carrier.
 10. A method of treating a patient suffering from a cancer expressing a death receptor 4, comprising administering a therapeutically effective dose of the pharmaceutical composition of claim 9 to the patient, thereby treating the cancer.
 11. The monoclonal antibody of claim 1, wherein the light chain CDRs comprise SEQ ID NOS:20, 21, and 22 respectively and the heavy chain CDRs comprise SEQ ID NOS:23, 24 and 25 respectively. 